This Pilot Project aims to identify the general and specific determinants of protein and ligand recognition in the ligand-binding domain (LBD) of nuclear receptors (NRs). Our focus will be the SF-1 and GCNF orphan NRs (ONRs), using an approach combining computational prediction of functional determinants followed by experimental validation through targeted mutagenesis. The functional assay system will be the regulation of Oct4 expression by SF-1 and GCNF during P19 cell differentiation. Our results will be a detailed map of the key functional residues of the SF-1 and GCNF LBDs, and will provide a general method applicable to other NRs. The Specific Aims are to: 1. Identify Key Functional Determinants on the LBDs of SF-1 and GCNF. We will apply the Evolutionary Trace (ET) to a diverse cross section of NR LBD sequences and, separately, to each member of the SF-1 and GCNF ONR sub- families. Similarities and differences between these analyses will allow us to predict key functional determinants shared by all LBDs, in addition to those specific for each family of orphans. 2. Validation of the ET on GCNF and SF-1 Using Directed Mutagenesis. We will mutate the residues identified in Aim 1 as being potentially functionally important within the LBDs of SF-1 and GCNF. These anticipated loss of function mutations will be tested in our Oct4 P19 cell expression system. We will assay for (i) loss of activation of SF-1, resulting in loss or decreased expression of Oct4, and (ii) loss of repression by GCNF leading to a failure to silence Oct4 expression.